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Methods in Pharmacology 
and Toxicology
Molecular 
Histopathology and 
Tissue Biomarkers in 
Drug and Diagnostic 
Development
Steven J. Potts
David A. Eberhard
Keith A. Wharton, Jr. Editors
M E T H O D S I N P H A R M A C O L O G Y
A N D T O X I C O L O G Y
Series Editor
Y. James Kang
Department of Medicine
University of Louisville School of Medicine
Prospect, Kentucky, USA
For further volumes: http://www.springer.com/series/7653
Molecular Histopathology
and Tissue Biomarkers
in Drug and Diagnostic
Development
Edited by
Steven J. Potts
Flagship Biosciences, LLC, Westminster, CO, USA
David A. Eberhard
University of North Carolina, Chapel Hill, NC, USA
Keith A. Wharton, Jr.
Novartis Institutes for BioMedical Research, Cambridge, MA, USA
Editors
Steven J. Potts
Flagship Biosciences, LLC
Westminster, CO, USA
Keith A. Wharton, Jr.
Novartis Institutes for BioMedical Research
Cambridge, MA, USA
ISSN 1557-2153 ISSN 1940-6053 (electronic)
Methods in Pharmacology and Toxicology
ISBN 978-1-4939-2680-0 ISBN 978-1-4939-2681-7 (eBook)
DOI 10.1007/978-1-4939-2681-7
Library of Congress Control Number: 2015939268
Springer New York Heidelberg Dordrecht London
# Springer Science+Business Media New York 2015
This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is
concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction
on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation,
computer software, or by similar or dissimilar methodology now known or hereafter developed.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not
imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and
regulations and therefore free for general use.
The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to
be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty,
express or implied, with respect to thematerial contained herein or for any errors or omissions that may have beenmade.
Printed on acid-free paper
Humana Press is a brand of Springer
Springer Science+Business Media LLC New York is part of Springer Science+Business Media (www.springer.com)
David A. Eberhard
University of North Carolina
Chapel Hill, NC, USA
Dedication
Chris Callahan, M.D., Ph.D.
This book is dedicated to the memory of our dear friend and esteemed colleague, Chris
Callahan, M.D., Ph.D., who suffered an untimely death from a progressive brain tumor in
2011. Already recognized as a leader early in his career, Chris held the position of Scientist
and Investigative Pathologist at Genentech/Roche at the time of his passing at the young
age of 46. Chris’s professional contributions pervade this book’s themes of molecular
histopathology and tissue-based biomarkers, and several of its contributors trained with or
worked alongside Chris at various stages of his career. More importantly, Chris represented
a generation of visionary physician-scientists who began their training in the 1980s, the last
“pre-genome” decade of human history, with the belief that the discovery of molecules and
pathways governing normal development would provide key insights into human disease.
While now considered dogma, at that time only scattered yet tantalizing hints existed to
indicate that alterations of these same handful of pathways, deeply conserved during our
evolution, caused (and were druggable targets of) diverse human diseases.
Chris completed his B.S. at Brown University in 1987, and earned his M.D. and Ph.D.
degrees as a graduate student in John Thomas’s lab at UC San Diego and Salk Institute
performing groundbreaking work in Drosophila neurobiology. His accomplishments
include the molecular cloning of derailed, a receptor tyrosine kinase crucial for axon
guidance [1]. Recognizing the central role of pathology in identifying molecular and
cellular mechanisms of disease, Chris moved to Stanford University to pursue residency
training in anatomic pathology, ultimately serving as Attending Physician and Acting
Assistant Professor of Pathology while engaged in postdoctoral research in Dermatology
with Tony Oro (also a former fellow M.D./Ph.D. student with Chris at UC San Diego).
Extending Tony’s postdoctoral work with Matthew Scott that implicated aberrant Hedge-
hog signaling in the most common human tumor, basal cell carcinoma [4], Chris went on
to discover a critical role for a Hedgehog pathway target gene Mtss1 (Missing in Metasta-
sis) in the regulation of signaling and cancer progression [3].
Tragically, Chris’s cancer diagnosis came at a most inopportune time—just as he
sought to start his own lab. Following aggressive surgery and chemotherapy, he returned
to the bench within days to continue his passion. By this time, Hedgehog pathway
v
inhibitors were being developed for oncology indications, and Chris felt the best opportu-
nity to apply his skills and talents to directly benefit cancer patients was to continue his
career as a pathologist in biopharma. Chris joined Genentech, supporting drug develop-
ment projects while directing several research projects aimed at understanding the mechan-
isms of Hedgehog pathway activation and therapy resistance in human cancer. Chris was a
key contributor to the R&D team efforts that led to the FDA approval of vismodegib, the
first-in-class Hedgehog pathway inhibitor for clinical use in advanced basal cell carcinoma.
The several hats Chris wore in these efforts included basic research and medical scientist,
translational biomarker and companion diagnostics R&D investigator, and pathology
advisor to the development team. Chris investigated cancer mechanisms to the end of his
life, with his final senior author paper on the role of Hedgehog signaling in tumor-stroma
interactions published in the Proceedings of the National Academy of Sciences just a few
months before his death [4].
Chris’s approach to life inspired those around him, as these quotes from two of his
Genentech colleagues attest:
(I have) never met anyone with more selfless dedication, engagement, focus, and commitment to his work
and family than Chris. Chris loved his role as a pathologist at Genentech and immersed himself in it 100 %. He
knew that his work was helping to transform the lives of patients, and felt fortunate that he could do so by
engaging in professional activities he loved most—basic hypothesis-driven research and scientific collaboration.
Chris knew, more clearly than most of us do, that his time with his family, his friends, and his work was
limited. He relished that time, and he shared it generously with others. Chris had a selfless, collegial
enthusiasm for his work. He was absolutely committed to his colleagues and to the projects he supported; he
would do everything he could to maximize the chances for their success. He had a great appreciation of, and
loved sharing subtle details of biology—not to advertise his brilliance, but because he trusted you would find
them as satisfying and wonderful as he did. When he dropped off his sons at school in the morning he would tell
them, ‘Have fun, learn a lot, and be kind.’ He fully modeled that advice.
Beyond describing Chris to a tee, these reminiscences illustrate three qualities of a
successful anatomic pathologist in biopharma that emerge as themes throughout this
book: a focus on the biology of disease, a passionate curiosity, and a collaborative mindset.
Although new insights about disease emerge daily, and opportunities for new discoveries
have never been greaterthan the present time, some things haven’t changed: A century and
half ago, Rudolf Virchow, founder of cellular pathology, said “If we would serve science,
we must extend her limits, not only as far as our own knowledge is concerned, but in the
estimation of others.” [5]. Chris exemplified this Virchowian ideal.
Comprehending the biology of disease requires integration of knowledge from diverse
disciplines, of which we concede the histopathology “stock in trade” of fixed and stained
tissue is only one part. As a role model, Chris excelled at the challenges faced by anatomic
pathologists embedded in drug and diagnostic industries, chief among them to bridge the
power (and limitations) of histopathology methods and knowledge with those from a
growing number of technology-driven disciplines, including genomics, protein biochem-
istry, quantitative image analysis, and in vivo imaging of cells to whole animals. Chris saw
firsthand that delivering a diagnostic test or new therapy to patients requires diverse skills,
far beyond what any one individual could possibly master. Whether performed in industry
or research institutes, drug development requires coordinated efforts by multidisciplinary
teams, and so the pathologist on the team must persuasively communicate with members
from diverse backgrounds and viewpoints in order to foster collaboration, and ultimately,
progress. Rare talents like Chris, with curious and creative minds, able to integrate
emerging data and knowledge across disciplines, are poised to see old problems in new
vi
ways and develop novel, important hypotheses that demand investigation. As Virchow said,
the pathologist must push the science—“. . .extend her limits. . .”—for all to see. Chris
fearlessly pursued multidisciplinary investigations in fruit flies, mice, and human systems in
order to understand core biologies and their alterations in human disease. We don’t yet
know if flies or mice will benefit from the fruits of Chris’s research, but humanity has
already benefited, and for that we are most grateful.
Chris and his wife Andrea are the proud parents of two boys, Nathan and Ryan.
Acknowledgements
Special thanks to Tony Oro, Cary Austin, and UCSD, Stanford, and Genentech colleagues
for their contributions to this dedication.
Cambridge, MA, USA Keith A. Wharton, Jr.
Chapel Hill, NC, USA David A. Eberhard
References
1. Callahan CA, Bonkovsky JL, Scully AL, Thomas JB (1996) derailed is required for muscle attachment
site selection in Drosophila. Development 122(9):2761–2767
2. Oro AE, Higgins KM, Hu Z, Bonifas JM, Epstein EH, Jr., Scott MP (1997) Basal cell carcinomas in
mice overexpressing sonic hedgehog. Science 276(5313):817–821
3. Callahan CA, Ofstad T, Horng L, Wang JK, Zhen HH, Coulombe PA, Oro AE (2004) MIM/BEG4,
a Sonic hedgehog-responsive gene that potentiates Gli-dependent transcription. Genes Dev 18
(22):2724-2729. doi:10.1101/gad.1221804
4. Chen W, Tang T, Eastham-Anderson J, Dunlap D, Alicke B, Nannini M, Gould S, Yauch R,
Modrusan Z, DuPree KJ, Darbonne WC, Plowman G, de Sauvage FJ, Callahan CA (2011) Canonical
hedgehog signaling augments tumor angiogenesis by induction of VEGF-A in stromal perivascular
cells. Proc Natl Acad Sci U S A 108 (23):9589-9594. doi:10.1073/pnas.1017945108
5. Virchow R (1858) Cellular pathology (trans: Chance F). Edwards Brothers, Inc., Ann Arbor, MI
vii
Preface
I’ve just sucked one year of your life away. . . What did this do to you? Tell me.
And remember, this is for posterity so be honest. How do you feel?
–Count Rugen, antagonist in the 1987 movie The Princess Bride
In the movie The Princess Bride, the hero, Westley, has just been subjected to The
Machine, a torture device that sucks years of life out of the victim. Like Westley, who cries
andmoans in pain in response to Count Rugen’s query, anyone embarking on, or reflecting
upon, a multi-year project knows the pathologic feeling of time spent on a lengthy and
complex project, whether it is a book, a drug, or a film.
Feature films aspiring for blockbuster status can consume $100 million or more in
production costs and 3 years just to get to production stage—all to entertain people for a
mere 2 hours. Yet this amount of money pales in comparison to the economic realities of
producing a new therapeutic that might address an unmet medical need for thousands or
even millions of people. By most measures, developing a new drug in 2015 costs at least ten
times more than a blockbuster movie. Three years in production is feature film fiction
compared to the industry average of ~14 years for drug development.
The feature film and the pharmaceutical industries face similar challenges: years
between the initial idea and a revenue-generating product, huge multifaceted teams,
millions of dollars invested in multiple projects, only a few of which succeed, and the
hope of the occasional blockbuster that must finance the failures of the rest.
At each phase in drug development, from early discovery through IND (Initial New
Drug Application) to NDA (New Drug Application), the promise of efficacy is balanced
against the penalty of toxicity. While there are many ways that efficacy and toxicity can be
evaluated in animals and in people, the highest concentration of information relevant to
many diseases remains the lesional tissue sample, microscopic examination of which pro-
vides a foundation to understand disease and the effect of therapy. Increasingly, whole
microscopic slide imaging is used, providing at least an order of magnitude higher resolu-
tion of cellular context than current noninvasive in vivo radiological imaging techniques.
However, microscopic data requires many players to extract its maximum value: histology
(preparing the tissue sample) and pathology (interpreting the tissue sample), in addition to
experts in disease-specific biology. Tissue-based studies help to understand how candidate
therapies act in animals and humans, and this work is often performed by small biotech
companies, large pharmaceutical companies, academic medical centers, commercial refer-
ence laboratories, and government entities. Each actor has a critical role to play in the
process and in the development of the final product.
We anticipate those who will most benefit from reading this book will be embedded in
government-sponsored academic research, diagnostics, or biopharmaceuticals, but we have
strived to make the chapters accessible and interesting to a wide audience. Due to shrinking
government budgets for basic research, more academic researchers are responding to grant
announcements and pharmaceutical partnerships that drive them deeper into drug devel-
opment. With the growth of companion diagnostics, experts in disease diagnostics will find
useful information in this volume about co-development of diagnostic and therapeutic
products, though the nature and timelines of the diagnostic industry are very different
from those of drug development, creating some unanticipated but, on deeper
ix
consideration, not so surprising challenges. Our analogy to the film industry provides
caution to those entering pharmaceutical drug development: While the biology underlying
drug development may be familiar—and thus appear simple—to those outside the bio-
pharma industry, one cannot overstate the complexities of drug development. One should
approach the study of the biopharma industry, and one important part of it—tissue
histopathology—with the same caution one might approach completely unfamiliar terri-
tory: with curiosity and respect for lessons learned by experience. Scientists from the
diagnostics industry are forewarned: while pharma and diagnostics have shared biology,
they are as dissimilar as are the pharma and moviemaking industries.
We often celebrate 2 hours of entertainment more than we give pause to acknowledge
medicines that positively impact humanlives. Recent progress in hepatitis C, cystic fibrosis,
and tumor immunology has been nothing short of astounding. Our industry can be the
best at times and the worst at times, but for many of us there is no more satisfying endeavor
than the opportunity to design therapeutics that have the potential to save and improve
lives.
It is our heartfelt belief that the biopharmaceutical industry makes positive and lasting
contributions to humanity. With the human genome completed just over a decade ago,
comparative genomics studies have revealed an array of druggable targets whose manipu-
lation is at the root of most therapy development programs today. In the not-so-distant
future, we are poised to witness dramatic improvements in the treatment of a myriad of
severe and debilitating diseases including infectious diseases, intractable and largely incur-
able cancers, as well as autoimmune and genetic diseases. Histopathology is central to this
effort, yet it is often relegated to a checkbox activity that is not given proper scrutiny or
thought. Our authors and editorial team, consisting of experts in histopathology, have
written from the trenches of diagnostic practice and pharmaceutical drug development,
aiming to educate pharmaceutical and academic scientists how to best use tissue in drug
development.
Most pathologists and histologists are, by their very nature, humble and not oriented to
marketing their wares. This book aims to help make their contributions to drug develop-
ment better understood as well as to identify best practices and new applications for their
trade. The book’s dedication highlights the contribution made by an exemplary
individual—a pathologist no longer in our midst—whose example continues to motivate us.
Audience
This book is intended for three audiences. First and foremost, it is written for all scientists
and managers in the Biopharma industry who must interact directly or indirectly with
tissue samples but whose primary training did not include pathology or other skills of tissue
interpretation; second, for pathology professionals and tissue scientists who will find some
of the examples of applications of their trade in drug development by their peers and
colleagues helpful; and third, for the many academic groups funded by government entities
to become more engaged in all stages of drug development.
x Preface
Information Content of a Tissue Biopsy
Both clinicians and the general public expect the pathologist’s interpretation to be the
“gold standard” of disease diagnosis—the absolute truth. “What were the path results?”
“Do I have cancer or not? What kind?” In most cases, pathological interpretation of a
relevant tissue sample is the final arbiter of truth. In both efficacy and toxicology studies,
there is much information to be gleaned from local tissue environment and context, with
the spatial and temporal characteristics of the cells in their organ preserved. While we strive
to link cell and tissue-level resolution with complex datasets that derive from -omics
analyses such as next-generation DNA and RNA sequencing, many forget that immuno-
histochemistry has provided single cell analysis of protein distribution—albeit a single
protein at a same time—for decades. Technology is always creating new approaches to
interrogate tissue samples, both within biopharma and in academia, but for a technology to
be implemented in clinical practice, it requires confirmation of utility and definition of its
limits and context that often comes years after the technology has lost its newness. But the
expectation that magically circulating in the blood is information content that will replace
the efficacy and toxicology information content of a tissue biopsy is a fantasy of Hollywood
proportions. Many people, even within the biopharma industry, are not aware that every
organ of every animal used in a GLP (Good Laboratory Practice) toxicology study must be
examined under the microscope by one or more veterinary pathologists. The FDA and
other regulatory agencies remain aware of the importance of the tissue microenvironment
to drug development.
How Important Is Histopathology in Drug
Development?
How do we assign an economic value to tissue analysis in drug development? One
approach is to estimate the number of slides generated or read per year, which then can
be converted to dollars. This can be estimated in several different ways, working from the
number of pathologists who engage in testing or the number of drugs that require testing
of tissue exposed to them. There are approximately 600 veterinary pathologists who work
primarily in pharmaceutical research, and probably another ~40 M.D. pathologists exclu-
sively working in pharmaceutical research. The vast majority of slides utilized in the
pharmaceutical industry are in support of GLP toxicological pathology studies for IND
submissions. Previous studies have estimated that on average one pathologist supports $1B
of drug product development [1]. We can assume each pathologist reviews ~10,000 slides
a year (~50 slides a day � ~200 working days a year) and yields an estimate of nearly ~7
million glass slides evaluated each year worldwide in drug development. However, this
number does not account for non-boarded pathologists and scientists who review slides, so
it may likely be closer to ten million glass slides per year. Even with a rough estimate of
$150 per glass slide for creation (histology) and reading (pathology), this equates to $1.5
billion spent annually on histopathology within the pharmaceutical industry.
Preface xi
Organization and Goals
While much of the focus of pathology in the biopharmaceutical industry is on oncology
programs, there is growing recognition of the value of pathology outside of oncologic
disease. Consequently, the chapters have been deliberately selected to include other disease
areas, including chapters addressing nonalcoholic steatohepatitis, arthritis, celiac disease,
myeloproliferative disorders, neurology, and wound healing. As part of a methods series,
this volume is designed to provide practical wisdom and examples that others can follow
and apply as part of drug development. Some chapters are case studies in specific techni-
ques or disease areas where tissue biopsies can be utilized effectively, while others are
literature reviews, and still others are a summary of the authors’ decades of collective
experience with tissue in an area of drug development.
While a great deal of pathology-related efforts in drug development are by necessity
geared toward formal toxicologic evaluation under GLP requirements, the editors have
deliberately not focused on toxicologic pathology as a discipline. This is certainly not
reflective of the relative importance of safety testing, but was rather for two other reasons:
First, toxicologic pathology in drug development has been comprehensively covered for
over a decade in a regularly updated excellent handbook for practitioners [2] as well as in
specialty journals. Second, the pace of change in toxicologic pathology is slow in compari-
son to other more technology-driven aspects of pathology commonly used in drug
development. The IND approval process allowing first-in-human studies is a highly regu-
lated endeavor, stipulated by GLP standards, that can take on a life (and a career) of its own.
Of necessity, innovation occurs slowly—and often reactively—in this field.
This book, which focuses on pursuits within industry that are variably termed “experi-
mental” or “investigative” pathology, or “translational medicine,” thus deals more with
efficacy studies that bridge from early stage discovery to formal clinical trials. An introduc-
tion to the field of anatomic pathology and its application to the biopharma industry is first
provided, based on the author’s experience both in industry and through teaching medical
students. This is followedby a personal narrative by a leading biopharma pathologist on the
nuances of communicating pathology results to non-pathologist colleagues, and then by a
chapter on planning and outsourcing histopathology-based investigations in clinical trials.
Two leading experts in inflammatory disease then provide a specific example of how
histopathology can be leveraged to better understand rheumatoid arthritis.
The second section focuses on applications of tissue image analysis–whole microscopic
slide imaging and computer algorithms to quantitatively measure what the pathologist
qualitatively observes. The chapters cover a variety of disease areas and concepts including
angiogenesis, hepatic fibrosis, and celiac disease, providing a glimpse of future applications
of digital pathology.
The third section discusses molecular histopathology, divided into in situ hybridization
(mRNA and DNA), sequencing, and genomics. The reader will find state-of-the-art
reviews with methodology on in situ hybridization as well as next-generation sequencing
of tissue samples.
The fourth section covers companion diagnostics. The first two chapters describe
preanalytic variables and then the adaptation of HER2 IHC scoring systems commonly
used in breast cancer to gastrointestinal tumors. Three chapters then discuss the develop-
ment of companion diagnostics from an industry standpoint, the relationship between the
xii Preface
reference lab, diagnostic partner and the pharma client, and regulatory aspects of medical
device submissions. A success story of a multianalyte IHC companion diagnostic is then
presented. Finally, two biostatisticians discuss statistical approaches to cut-point analysis in
digital pathology and applications in IHC companion diagnostics.
We hope that this volume will serve to inform and enlighten both tissue-focused and
non-tissue-focused drug development scientists about better use and interpretation of the
multidimensional data contained in a tissue biopsy. The hunt for new therapies remains one
of the most exciting and meaningful pursuits in the twenty-first century, and so the
evaluation of a tissue biopsy remains a central and challenging part of that pursuit.
Westminster, CO, USA Steven J. Potts
Cambridge, MA, USA Keith A. Wharton, Jr.
Chapel Hill, NC, USA David A. Eberhard
References
1. Potts SJ, Young GD, Voelker FA (2010) The role and impact of quantitative discovery pathology.
Drug Discov Today 15(21–22): 943-50
2. Haschek WM, Rousseaux CG, Wallig MA (2002) Handbook of toxicologic pathology, Vol 1.
Academic, San Francisco
Preface xiii
Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
Dedication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xvii
Histopathology: A Canvas and Landscape of Disease in Drug
and Diagnostic Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Keith A. Wharton Jr
A Field Guide to Homo morphologicus for Biomedical Scientists,
Or How to Convey an Understanding of Pathology to Scientists
in a Biopharma Enterprise . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Humphrey Gardner
Outsourcing Tissue Histopathology Investigations in Support
of Clinical Trials for Novel Therapeutics: Considerations and Perspectives . . . . . . . . 43
Keith A. Wharton Jr., Benjamin H. Lee, Pierre Moulin, Dale Mongeon,
Rainer Hillenbrand, Arkady Gusev, Bin Ye, and Xiaoyu Jiang
Histopathology in Mouse Models of Rheumatoid Arthritis . . . . . . . . . . . . . . . . . . . . . 65
Patrick Caplazi and Lauri Diehl
Markers Used for Visualization and Quantification of Blood
and Lymphatic Vessels. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
Mohamed E. Salama, David A. Eberhard, and Steven J. Potts
Practical Approaches to Microvessel Analysis: Hotspots,
Microvessel Density, and Vessel Proximity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
Steven J. Potts, David A. Eberhard, and Mohamed E. Salama
Quantitative Histopathology and Alternative Approaches to Assessment
of Fibrosis for Drug Development in Hepatitis C and Nonalcoholic
Steatohepatitis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
Steven J. Potts and Johanna K. DiStefano
Stereology and Computer-Based Image Analysis Quantifies Heterogeneity
and Improves Reproducibility for Grading Reticulin in Myeloproliferative
Neoplasms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
Mohamed E. Salama, Erik Hagendorn, Sherrie L. Perkins, Jeff L. Kutok,
A. Etman, Josef T. Prchal, and Steven J. Potts
Image Analysis Tools for Quantification of Spinal Motor Neuron
Subtype Identities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
Mirza Peljto and Hynek Wichterle
Development of a Tissue Image Analysis Algorithm for Celiac
Drug Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
Erik Hagendorn, Christa Whitney-Miller, Aaron Huber,
and Steven J. Potts
Quantitative Histopathology for Evaluation of In Vivo Biocompatibility
Associated with Biomedical Implants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
Robert B. Diller, Robert G. Audet, and Robert S. Kellar
xv
Quantitative Histomorphometry and Quantitative Polymerase Chain
Reaction (PCR) as Assessment Tools for Product Development . . . . . . . . . . . . . . . . . 163
Robert G. Audet, Robert B. Diller, and Robert S. Kellar
Measuring the Messenger: RNA Histology in Formalin-Fixed Tissues. . . . . . . . . . . . 175
Steven J. Potts, Mirza Peljto, Mahipal Suraneni, and Joseph S. Krueger
Algorithm-Driven Image Analysis Solutions for RNA ISH Quantification
in Human Clinical Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
Mirza Peljto, Joseph S. Krueger, Nicholas D. Landis, G. David Young,
Steven J. Potts, and Holger Lange
Solid Tissue-Based DNA Analysis by FISH in Research and Molecular
Diagnostics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
Marcus Otte
Preanalytic Considerations for Molecular Genomic Analyses of Tissue. . . . . . . . . . . . 203
Maureen Cronin
Next-Generation Sequencing (NGS) in Anatomic Pathology Discovery
and Practice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219
Matthew J. McGinniss, David A. Eberhard, and Keith A. Wharton Jr
The Impact of Pre-analytic Variables on Tissue Quality from Clinical
Samples Collected in a Routine Clinical Setting: Implications
for Diagnostic Evaluation, Drug Discovery, and Translational
Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259
David G. Hicks
Adapting HER2 Testing for a Different Organ: New Wine
in Old Wineskins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271
Michael D. Lunt and Christa L. Whitney-Miller
Tissue-Based Companion Diagnostics: Development of IHC Assays
from an Industry Perspective . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . 281
Miu Chau and Jon Askaa
Development of Tissue-Based Companion Diagnostics:
The Relationship Between the Pharmaceutical Company,
Diagnostic Partner, and the Biomarker Laboratory . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305
Mark Kockx, Stefanie de Schepper, and Christopher Ung
Navigating Regulatory Approval for Tissue-Based Companion Diagnostics . . . . . . . 325
Joseph S. Krueger, Holger Lange, G. David Young, and Steven J. Potts
Implementing a Multi-analyte Immunohistochemistry Panel
into a Drug Development Program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345
Carla Heise, Pierre Brousset, Tommy Fu, David A. Eberhard,
Graham W. Slack, Camille Laurent, and Randy D. Gascoyne
Cutpoint Methods in Digital Pathology and Companion Diagnostics . . . . . . . . . . . . 359
Joshua C. Black, Mahipal V. Suraneni, and Steven J. Potts
Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373
xvi Contents
Contributors
JON ASKAA � Medical Prognosis Institute, Hørsholm, Denmark
ROBERT G. AUDET � Development Engineering Sciences LLC, Flagstaff, AZ, USA
JOSHUA C. BLACK � Flagship Biosciences, Westminster, CO, USA
PIERRE BROUSSET � Department of Pathology, CHU Toulouse-Purpan, Toulouse, France
PATRICK CAPLAZI � Department of Research Pathology, Genentech, Inc., South San Francisco,
CA, USA
MIU CHAU � Genentech, Inc., South San Francisco, CA, USA
MAUREEN CRONIN � Strategic Information Management, Celgene Corporation, San
Francisco, CA, USA
LAURI DIEHL � Department of Research Pathology, Genentech, Inc., South San Francisco,
CA, USA
ROBERT B. DILLER � Development Engineering Sciences LLC, Flagstaff, AZ, USA;
Department of Biological Sciences, Center for Bioengineering Innovation, Northern
Arizona University, Flagstaff, AZ, USA
JOHANNA K. DISTEFANO � TGEN, Phoenix, AZ, USA
DAVID A. EBERHARD � Department of Pathology and Laboratory Medicine, Department
of Pharmacology and Lineberger Comprehensive Cancer Center, University of North
Carolina, Chapel Hill, NC, USA; Laboratory Corporation of America (LabCorp),
Research Triangle Park, NC, USA
A. ETMAN � University of Utah & ARUP Laboratories, Salt Lake City, UT, USA
TOMMY FU � Celgene Corporation, Summit, NJ, USA
HUMPHREY GARDNER � Translational Medicine, Early Clinical Development, AstraZeneca
R&D, Waltham, MA, USA
RANDY D. GASCOYNE � Department of Pathology and the Center for Lymphoid Cancer,
Organization British Columbia Cancer Agency, Vancouver, BC, Canada
ARKADY GUSEV � Biomarker Development, Translational Medicine, Novartis Institutes
for BioMedical Research, Cambridge, MA, USA
ERIK HAGENDORN � Flagship Biosciences, Westminster, CO, USA
CARLA HEISE � Celgene Corporation, Summit, NJ, USA
DAVID G. HICKS � Surgical Pathology Unit, Department of Pathology and Laboratory
Medicine, University of Rochester Medical Center, Rochester, NY, USA
RAINER HILLENBRAND � Biomarker Development, Translational Medicine, Novartis
Institutes for BioMedical Research, Basel, Switzerland
AARON HUBER � School of Medicine and Dentistry, University of Rochester, Rochester, NY,
USA
XIAOYU JIANG � Biomarker Development, Translational Medicine, Novartis Institutes
for BioMedical Research, Cambridge, MA, USA
ROBERT S. KELLAR � Development Engineering Sciences LLC, Flagstaff, AZ, USA;
Department of Biological Sciences, Center for Bioengineering Innovation, Northern
Arizona University, Flagstaff, AZ, USA; Department of Mechanical Engineering, Center
for Bioengineering Innovation, Northern Arizona University, Flagstaff, AZ, USA
MARK KOCKX � HistoGeneX, Antwerp, Belgium
JOSEPH S. KRUEGER � Flagship Biosciences, Westminster, CO, USA
xvii
JEFF L. KUTOK � Infinity Pharmaceuticals, Inc., Cambridge, MA, USA
NICHOLAS D. LANDIS � Flagship Biosciences, Westminster, CO, USA
HOLGER LANGE � Flagship Biosciences, Westminster, CO, USA
CAMILLE LAURENT � Department of Pathology, CHU Toulouse-Purpan, Toulouse, France
BENJAMIN H. LEE � Oncology Translational Medicine/Oncology Business Unit, Novartis
Institutes for BioMedical Research, Cambridge, MA, USA
MICHAEL D. LUNT � School of Medicine and Dentistry, University of Rochester, Rochester,
NY, USA
MATTHEW J. MCGINNISS � Medical Laboratory, Genoptix, a Novartis Company, Carlsbad,
CA, USA
DALE MONGEON � Biomarker Development, Translational Medicine, Novartis Institutes
for BioMedical Research, Cambridge, MA, USA
PIERRE MOULIN � Discovery and Investigative Pathology, Preclinical Safety, Novartis
Institutes for BioMedical Research, Basel, Switzerland
MARCUS OTTE � Oridis Biomarkers, Graz, Austria
MIRZA PELJTO � Flagship Biosciences, Westminster, CO, USA
SHERRIE L. PERKINS � University of Utah & ARUP Laboratories, Salt Lake City, UT, USA
STEVEN J. POTTS � Flagship Biosciences, Westminster, CO, USA
JOSEF T. PRCHAL � University of Utah & ARUP Laboratories, Salt Lake City, UT, USA
MOHAMED E. SALAMA � Department of Pathology, ARUP Reference Lab Research Institute,
University of Utah, Salt Lake City, UT, USA
STEFANIE DE SCHEPPER � Immunohistochemistry, HistoGeneX, Antwerp, Belgium
GRAHAM W. SLACK � Department of Pathology and the Center for Lymphoid Cancer,
Organization British Columbia Cancer Agency, Vancouver, BC, Canada
MAHIPAL V. SURANENI � Flagship Biosciences, Boulder, CO, USA; Flagship Biosciences,
Westminster, CO, USA
CHRISTOPHER UNG � HistoGeneX, Antwerp, Belgium
KEITH A. WHARTON JR. � Discovery and Investigative Pathology, Preclinical Safety, Novartis
Institutes for BioMedical Research, Cambridge, MA, USA
CHRISTA L. WHITNEY-MILLER � School of Medicine and Dentistry, University of Rochester,
Rochester, NY, USA
HYNEK WICHTERLE � Department of Pathology, Columbia University,
New York, NY, USA; Department of Neurology, Columbia University, New York, NY,
USA; Department of Neuroscience, Columbia University, New York, NY, USA
BIN YE � Biomarker Development, Translational Medicine, Novartis Institutes for
BioMedical Research, Cambridge, MA, USA; Beijing Shenogen Biomedical Co., Beijing,
China
G. DAVID YOUNG � Flagship Biosciences, Westminster, CO, USA
xviii Contributors
Methods in Pharmacology and Toxicology (2015): 1–26
DOI 10.1007/7653_2014_33
© Springer Science+Business Media New York 2014
Published online: 22 January 2015
Histopathology: A Canvas and Landscape of
Disease in Drug and Diagnostic Development
Keith A. Wharton Jr.
Abstract
The aims of diagnostic and therapeutic development are to accurately diagnose and cure disease,
respectively. For the past century and a half, histopathology—the microscopic examination of cells and
tissues—has been considered a “gold standard” for the diagnosis of many diseases. As an introduction
to this volume on molecular histopathology and tissue biomarkers in drug and diagnostic development,
I explore the relationship between histopathology and the nature of disease itself. A lack of agreement on
the meaning of “disease” has led to widespread and indiscriminate use of the term. Here, I propose that the
term “disease” be reserved for conditions where there exists some knowledge of alterations in cells or their
products that participate in cause-effect relationships in lesional (diseased) tissue. This is a definition that
simultaneously lends legitimacy to the term’s use while enabling revision and testing of hypotheses based on
rapidly emerging scientific knowledge. With this perspective, histopathology, as a preferred means to
visualize and depict the cellular events that constitute disease as it impacts tissue structure and function,
will remain essential to develop new diagnostic tests and targeted therapies for the foreseeable future.
Key words Disease, Histopathology, Biomarker, Lesion, Diagnosis, Illness, Condition,Disorder,
Pathogenesis, Feedback, Crosstalk, Clinical trial, Diagnostic test, Drug development
1 Introduction
If thought corrupts language, language can also corrupt thought.
—George Orwell
I rob banks because that’s where the money is.
—Willie Sutton
The concept of “disease” is central to understanding health in all
forms of life as well as for optimal delivery of health care. All human
cultures have words or symbols that represent disease or related
ideas. From our earliest experiences, we develop an understanding
of its general nature, but the term is increasingly adapted to suit
circumstances at hand. Diagnostic criteria, a set of features that
define each disease, range from simple to complex. For some well-
understood diseases, diagnosis is clear-cut, relying on defined and
measurable criteria. For poorly understood or controversial
1
diseases, observation and professional judgment play a major role,
creating opportunities for uncertainty, bias, and misdiagnosis.
Health care providersmake treatment decisions and bill for reimbur-
sements based on an incomplete and evolving understanding of
many diseases. Perusal of http://www.clinicaltrials.gov/, the USA
FDA’sWeb site thatdiscloses the essentials of all humanclinical trials,
reveals that enrollment criteria and response variables (i.e., trial end-
points) formanydiseases consist of features learned from themedical
history, or of clinical observations andmeasurements with uncertain
relevance to the disease processes under investigation. To the
afflicted individual, having a particular disease can alter self-identity
and behavior, and can impart social stigma and/or advantage.
Disease, frankly, is a big deal. Given that shared goals of biomedical
research, the biopharmaceutical industry, and the health care indus-
try as a whole are accurate diagnosis and treatment of disease, as an
introduction to this timely book devoted to molecular histopathol-
ogy and tissue biomarkers in drug and diagnostic development, it
seems reasonable to first consider the nature of disease itself.
Now, early in the twenty-first century, humanity finds itself
squarely in the middle of the molecular era of disease, full of
optimism that, from our recently sequenced genomes, cures for
our myriad diseases are imminent. Investigations from diverse fields
of biomedicine have revealed over the past 50 or so years the
molecular underpinnings, or at least a framework, for thousands
of hitherto mysterious and sometimes misclassified diseases—
spanning from the rare and precisely defined to the common and
heterogeneous. For example, there are at least 456 distinct genetic
diseases that affect the skeletal system [1], a majority for which
causative gene mutations have been identified. We have learned that
the culprits in many human (and animal) diseases are corrupted
versions of vital processes deeply conserved in our evolutionary
history—alterations in specific proteins, protein complexes, macro-
molecular structures, and organelles that comprise molecular com-
munication pathways with developmental, homeostatic, and
adaptive functions. In some cases, we know which cells misbehave
and how, and we can replicate key features of the disease in wild-
type or genetically altered animals. Since organisms are composed
of massive networks of interconnected, compartmentalized molec-
ular pathways, the task that remains for each disease, in each
patient, is to understand the dynamic, physical nature of each
pathway alteration and then devise strategies to oppose the abnor-
mal network of behaviors that promote disease without
compromising functions of the remaining pathways required for
life.
2 Keith A. Wharton Jr.
2 What Is Histopathology?
Histopathology is the microscopic visualization of tissue to describe
how cellular and tissue morphology is altered by manifestations of
disease (http://en.wikipedia.org/wiki/Histopathology). It is dis-
tinct from techniques that use (and destroy) tissue as a raw material
for nucleic acid sequencing or bioassays to measure specific mole-
cules or their concentrations, each of which separate the data
generated from its endogenous tissue context. Histopathology
thus provides a visual, descriptive picture of cells and tissues, fixed
or frozen in time, in their natural context—a landscape of sorts.
When viewed using the microscopic techniques of histopathology,
normal tissue might resemble a well-ordered city when viewed from
the sky, whereas tissue from a destructive autoimmune disease
might look like a war zone, and a cancer might kindle images of
prisoners escaping Alcatraz or Godzilla tipping skyscrapers. Histo-
pathology is central to patient care, whether to discern normal
tissue from benign or malignant disease, to ascertain surgical tissue
margins, or to investigate cause of death. Among all diagnostic
assays, histopathology remains unparalleled in its ability to discrim-
inate among a seemingly infinite variety of disease states, especially
in the presence of a much greater excess of normal tissue. For
example, in an appropriate biopsy, a trained pathologist can diag-
nose prostate cancer by recognizing less than ten malignant
(cancerous) cells among thousands of nonmalignant cells. Histopa-
thology is also a crucial tool to assess efficacy and possible safety
hazards of candidate human therapies in preclinical (i.e., animal)
studies. Despite the regulatory requirement for histopathology-
based interpretation of tissues in animal toxicology studies, collect-
ing tissues as part of human clinical trials remains a rare practice,
except in oncology trials. (A later chapter in this book explores
issues to consider when incorporating tissue and histopathology-
based measurements in human clinical trials.)
Anatomic pathologists are the sages of histopathology. They
are mostly physicians (M.D., D.O., or equivalent) or veterinarians
(D.V.M. or equivalent) who have completed one or more anatomic
pathology fellowships and often have research experience in inves-
tigative pathology or related scientific disciplines. They train over
years and decades to render interpretations and diagnoses based on
observable and, to an outsider, sometimes arcane criteria. Despite
the fact that Pathology, translated from its Greek origins, literally
means the study of suffering, pathology emerged as a medical
specialty, in part, to provide an objective diagnosis of disease, unbi-
ased by patient presentation or a physician’s subjective (but often
crucial) knowledge about a patient’s history. While a computer,
IBM’s Watson, can win the TV game show Jeopardy, no computer
is yet capable of replacing a diagnostic anatomic pathologist.
Histopathology: A Canvas and Landscape of Disease in Drug and Diagnostic Development 3
In this book, the editors and contributors adopt a far broader
view of histopathology, to include any discipline or technique that
enhances our ability to visualize and quantify the three-
dimensional, physical nature of normal and diseased tissue, includ-
ing advanced techniques in microscopy and cell biology, image
segmentation and quantitation, omics-scale profiling of various
molecular species, and “big data” analysis. Thus histopathology is
not a static or archaic art based only on routine stains of tissue
sections, but rather highly technical and continually evolving.
These ancillary technologies thus add more, and often critical,
detail to the landscape that is each disease. Modern histopathology
is a firmly molecular pursuit, encompassing the detection of specific
proteins by immunohistochemistry (IHC) and nucleic acids by in
situ hybridization (ISH). The editors espouse the view that data
generated from techniques that destroy tissue must be interpreted
in the context of methods such as histopathology that preserve it, in
order to best understand the manifestations of each diseasein tissue
and in the patient. Modern histopathology thus requires patholo-
gists to collaborate with a wide variety of scientists and engineers
who generate complementary, often deeper insights from diseased
tissue, in order to create a coherent working model of each disease
process.
3 What Is Disease?
Historical concepts of disease arose independently in diverse civili-
zations, including causation by external factors (e.g., gods, demons,
spells, miasmas) and imbalances in internal qualities (e.g., the four
humors). Largely incorrect in their primitive forms, each view has
elements of truth: disease can be initiated by external factors (e.g.,
infectious agents, toxin exposure) and can originate or manifest as
imbalances in internal qualities (e.g., persistent hormone produc-
tion or accumulation of toxic metabolites, mutations, or rogue
cells). Historical review of disease conceptions across cultures is
outside the scope of this chapter, but several sources exist [2–4].
Clinical acumen and technology jointly drive the discovery and
diagnosis of disease. Many diseases are—or were, at some point in
history—named for the person, usually a physician, who first
noticed a nonrandom aggregation of clinical or histopathological
features in their patients; Alois Alzheimer and James Parkinson are
two whose namesakes persist, though many names of diseases,
proper and otherwise, have since been discarded in favor of more
descriptive designations. The light microscope revealed fine details
of healthy and diseased tissues in the mid-nineteenth century,
revolutionizing disease diagnosis, classification, and nomenclature.
Thousands of discrete disease entities are recognized by the Inter-
national Classification of Diseases, ninth revision (ICD-9), a list
4 Keith A. Wharton Jr.
subject to revision every few years based on emerging knowledge
and expert consensus. Recognize, however, that such lists are made
largely for the purposes of counting and billing, and their compo-
sition usually lags behind the most current thinking about disease.
Today, a panoply of omics techniques (genomics, proteomics, etc.)
and in vivo imaging modalities give unprecedented insight into the
complexity of disease as it affects our bodies; these technologies
challenge prevailing notions of disease, force continual reassess-
ment of naming conventions, and can allow earlier, more accurate
diagnosis and monitoring of disease. For example, diagnosis of
Alzheimer’s dementia, in the past requiring an autopsy, can now
be made premortem with some certainty by combining cognitive
testing with imaging and cerebrospinal fluid biomarker analysis [5].
Massively parallel (next generation) sequencing, the subject of
another chapter in this book, stands to revolutionize disease diag-
nosis, classification, and monitoring in the next decade. Principles
of systems biology are being applied to datasets derived from
diseased tissue or fluids in an attempt to better classify, understand,
and treat disease (e.g., ref. 6). Such studies create compelling and
often testable hypotheses. However, the enormous sizes of datasets
in comparison to the typically small numbers of diseased samples
used to generate them have raised concern that resulting predictive
models will be overfitted to the diseased sample set and thus not
applicable to the population as a whole.
Many consider “disease” a more specific concept than simply
the absence of health, and defining it, in general and for each
specific case, is not a trivial exercise. One classic definition of
disease, articulated by Sir John Russell Reynolds in 1866, is: “any
condition of the organism which limits life in either its power, enjoy-
ment, or duration” [7]. Merriam Webster (www.m-w.com) defines
disease as, “a condition of the living body or of one of its parts that
impairs normal functioning and is typically manifested by distin-
guishing signs and symptoms.” (Recall that symptoms are experienced
by the patient, such as insomnia or pain, whereas signs are observed
by the physician during physical examination; e.g., Courvoisier’s
sign is a palpable gallbladder in a patient with obstructive jaundice).
Wikipedia (c. 2014) offers a wider, more inclusive definition:
“. . .any condition that causes pain, dysfunction, distress, social pro-
blems, or death to the person afflicted, or similar problems for those in
contact with the person. In this broader sense, it sometimes includes
injuries, disabilities, disorders, syndromes, infections, isolated symp-
toms, deviant behaviors, and atypical variations of structure and
function.” Unfortunately, here inclusion breeds imprecision,
although given the mutable nature of Wikipedia the definition
might be changed by the time you read this chapter!
Such definitions of disease, both classic and modern, are inade-
quate tools for thinking about and communicating modern con-
cepts of disease. Too often language, entrenched by historical
Histopathology: A Canvas and Landscape of Disease in Drug and Diagnostic Development 5
accidents of discovery, shapes the boundaries of our thought, when
thought, shaped by scientific discovery, should enhance the accu-
racy and precision of language that describes the products of our
thought. These observations suggest a new working definition of
disease is needed—one that not only reflects scientific principles
upon which experts can agree, but that also facilitates thinking
about new ways to treat or cure disease. This (or any) attempt to
create a single umbrella definition for disease might be regarded as
futile, but one hopes the effort helps to focus scrutiny on disease-
relevant events.
4 Three Concepts Related to Disease
Disease can be considered distinct from illness, which m-w.com
defines as “unhealthy in body or mind,” but commonly refers a
patient’s subjective experience of disease. Given that one can feel
ill without having a known disease or have a severe disease with no
ill feelings (e.g., early stage cancer), it is reasonable to assume that a
defined disease need not be a prerequisite for illness, or at least
temporarily ill feelings. Treating symptoms of disease, illness, and
suffering are laudable goals, but the view of disease proposed here,
absent knowledge about the disease process itself, might not inform
the best way to manage illness. (Note that alternate definitions of
“illness” are nearly synonymous with disease).
Disease is distinct from a medical condition, which implies
deviation from normalcy but is not necessarily an abnormality.
For example, pregnancy is a temporary condition, but not an
abnormal one. Disease is also distinct from a disorder, a term related
to disease but with the implication that much less is known about
the nature of what’s wrong. Disorder is often used to describe
intrinsic abnormalities of brain function. For example, the Diag-
nostic and Statistical Manual of Mental Disorders (DSM), currently
in its fifth revision, is an ongoing but imperfect attempt to classify
disorders of thought, feeling, or behavior. A few entities described
in the current DSM qualify as a “disease” by the standards of this
chapter (e.g., those conditions highly associated with mutations in
genes that act in known pathways impacting specific regions of the
brain), but their mysterious nature precludes classification by cri-
teria more precise than those based on constellations of behavior or
symptoms. Disorder is also used as a term that refers to a family of
diseases or their manifestations, e.g., disorders of metabolism.
Designation as “disease” remains controversial and is not with-
out consequence. Ill feelings, a condition, or even normal physio-
logical variation can be “medicalized” or “pathologized” into
disease bymotivated parties, including scientists seeking grant fund-
ing or biopharmaceutical companies seeking a new market niche.
For example, thereis ongoing debate whether certain life-cycle-
6 Keith A. Wharton Jr.
associated conditions are bona fide diseases or are simply conse-
quences of aging that many in Western society refuse to accept.
Companies that make products such as nutritional supplements
not subject to FDA approval craft language to imply that their
product is beneficial (“supports the health of. . .” is a commonly
used phrase) for this or that organ (and by implication, will oppose
disease of that organ) while required byUSA federal law to state that
their “product is not intended to diagnose, treat, cure, or prevent any
disease.” Carrying a diagnosis of a so-named disease has socioeco-
nomic consequences. The American Medical Association’s recent
declaration that obesity is a disease drew attention to its increasing
prevalence and impact on society’s health [8], but at the same time
affixed a label of questionable utility beyond possession of a “risk
factor” for other diseases such as diabetes, hypertension, and
myocardial infarction to millions of overweight people. Often,
implicit in affixing the label of “disease” is absolution of blame
for the affected individual’s state of health or actions resulting
from it. Addiction, personality disorders, or willful criminal acts:
are they diseases or not? Any debate about the nature of disease can
quickly transcend science, reaching the realm of philosophy and
raising the question: Do we control the disease or does the disease
control us? Highlighting our pervasive inability to agree on what
constitutes disease, an insightful New Yorker magazine article [9]
quotes E.M. Jellinek’s mid-twentieth century work that advocated
alcoholism as a disease: “A disease is what the medical profession
recognizes as such.”
Given the stakes, there is need for a working definition of
disease that simultaneously lends credibility to use of the term,
while enabling revision of disease concepts based on emerging
scientific knowledge. I propose that bona fide “disease” be consid-
ered an abnormality of the organism, caused by abnormal cells or
their products, leading to cause-effect relationships in lesional tissue
that compromise function in a societal context or attenuate normal
lifespan. Through visualization of diseased cells and tissues, histo-
pathology becomes both the canvas and the landscape upon which
disease unfolds.
5 Organic Disease vs. Functional Disease
In line with the proposed definition of disease, the medical profes-
sion has long distinguished between organic and functional disease.
Organic disease can cause a measurable physiological change, and
thus changes in molecules, cells, tissues, or organs that give rise to
the disease (or for which such changes are a consequence of the
disease) can be identified and often measured with a diagnostic test.
Functional diseases, sometimes called functional symptoms or dis-
orders, can manifest as signs, symptoms, or somehow altered
Histopathology: A Canvas and Landscape of Disease in Drug and Diagnostic Development 7
function (e.g., pain, fatigue, or other form of suffering) without
apparent alterations in known tests. As science progresses, many
functional disorders will join the ranks of organic diseases.
For example, “inflammatory bowel disease” encompasses several
organic diseases of the bowel in which alterations in tissue structure
documented by histopathology generally correlate with the severity
of clinical symptoms, whereas “functional bowel disease” (a.k.a.
“irritable bowel syndrome”), despite sometimes equally debilitat-
ing symptoms, is characterized by an absence of specific abnormali-
ties by histopathology, but has been associated with clinical
depression. The organic/functional distinction can become blurred
in certain instances: although we do not know exactly which cells
are altered, and how, in the brains of patients with schizophrenia or
autism, few doubt their organic nature. Some disorders currently
classified as functional are associated with psychological or psychi-
atric conditions, raising the question of whether they are somatic
(body) manifestations of an underlying psychiatric condition or
disorder. Pathologists claim dominion over organic disease, leaving
the more difficult questions of functional disease and its manage-
ment to other specialists. A diagnosis of exclusion is one that is made
after other causes are excluded: some diseases, like the multisystem
granulomatous disease sarcoidosis, are clearly organic, whereas
others not currently linked to definitive test results are classified as
functional but may be no less a burden than organic disease to those
afflicted.
6 Three More Concepts Related to Disease
Three more concepts deserve mention before I further examine the
nature of disease.
A syndrome is a nonrandom association of several apparently
unrelated features (signs, symptoms, test results) that suggest a
common underlying cause or pathogenesis. Down syndrome, a
constellation of developmental defects due to trisomy 21, and
Acquired Immune Deficiency Syndrome (AIDS), caused by HIV
infection-induced immune deficiency, are classic examples.
Pathognomonic describes the almost certain presence of a disease
(or disorder) when a particular sign or symptom, or a combination
thereof, is present, or with a particular diagnostic test result. In
statistical terms, this means that the “positive predictive value” (the
percentage of patients with a feature such as a positive test result that
have thedisease) of a particular sign, symptom,or test result is 100%.
Two skin diseases illustrate these two points. Cutaneous
xanthomas (small skin papules or nodules composed predomi-
nantly of macrophages stuffed with cholesterol) are pathogno-
monic for hyperlipoproteinemia; i.e., all individuals with
xanthomas have some sort of alteration in lipoprotein metabolism.
8 Keith A. Wharton Jr.
Dozens to hundreds of cutaneous basal cell carcinomas in sun-
exposed areas on a young adult are pathognomonic for Gorlin’s
basal cell nevus syndrome, caused by a heterozygous, germ line
mutation in the PTCH1 gene [10]. But, how specific the relation-
ship is between the pathognomonic feature and the disease varies
with the definition and cause of each disease: not all individuals with
hyperlipoproteinemias have xanthomas, and there are many differ-
ent causes of hyperlipoproteinemia. In contrast, the relationship
between PTCH1 mutation and Gorlin’s syndrome is obligate, in
that there appears to be no other genes that when singly mutant
give rise to such a specific, striking array of clinical manifestations.
The third concept is forme fruste, which describes a very mild or
atypical form of a disease. One example is discoid lupus, a variant of
the autoimmune disease systemic lupus erythematosus (SLE)
whose manifestations are restricted to skin. Sometimes, a forme
fruste variant manifests in a distinct tissue or organ compared to the
more typical or severe form of the disease. For example, cystic
fibrosis, due to mutation in the CFTR channel protein, has severe,
life-threatening manifestations in the lung and pancreas, but occa-
sional patients with partial loss of function mutations in the same
protein present with male infertility due to the requirement of
CFTR for vas deferens function [11]. Hence, male infertility can
be a forme fruste of cystic fibrosis. Since each of us harbors on the
order of 400 defective genes in our genomes and inherits ~60 new
mutations not present in our parents’ DNA [12, 13], widespread
use of massively parallel sequencing technologies in future diagnos-
tic tests (the subject of another chapter in this book) is likely to
reveal combinations of sequence polymorphisms or otherwise cryp-
tic gene mutations as factors contributing to “forme frustes” of
many diseases.
7 Etiology/Chain of Causation
The foundation of differential diagnosis—the processof determin-
ing which among a list of potential diseases a patient might have
based on their unique presentation—is classification of disease by
apparent cause or etiology. Broad, descriptive categories have tradi-
tionally been used to classify disease: these include neoplastic,
infectious, genetic, vascular, and idiopathic—the latter signifying
our ignorance of an underlying cause. Distinctions are made
between congenital and acquired disease, and genetic, familial,
and sporadic disease; these categories will not be further explored.
Diseases, however (as defined), can be considered primary if they
originate within the named organ, or secondary or even tertiary if
causative factors originate from other organs. For example, primary
hyperparathyroidism is caused by autonomous hypersecretion of
parathyroid hormone (PTH) from the parathyroid gland (e.g., by a
Histopathology: A Canvas and Landscape of Disease in Drug and Diagnostic Development 9
parathyroid cell-derived tumor or from nonmalignant parathyroid
cells harboring a mutation that activates a signaling pathway, lead-
ing to PTH hypersecretion). Since the function of PTH is to
increase serum calcium by acting on a variety of target tissues
(bone, kidney, intestine), secondary hyperparathyroidism is charac-
terized by increased parathyroid secretion from the parathyroid
gland, usually due to the body’s attempt to correct for low serum
calcium. The manifestations of secondary hyperparathyroidism on
target organs outside of the parathyroid gland are similar to those
of primary hyperparathyroidism, yet the causes are very different.
Sometimes, a presumptive or working diagnosis of disease is
made if time is limiting and the consequences of missing a diagno-
sis, and the appropriate intervention, are dire. For example, pneu-
monia is commonly treated with medical therapy based on clinical
history and symptoms, often confirmed with an X-ray, but without
identification of a causative infectious agent. Even a definitive diag-
nosis of a disease in a given patient should inspire further inquiry,
sometimes urgently. For example, while iron deficiency anemia can
be considered a disease—characterized by abnormal test results,
signs, and symptoms—its presence beckons the search for a cause.
Indeed, distinct, qualitative categories of disease, coupled with
ordered (e.g., primary and secondary) events during disease pro-
gression, implies that a sequence of discrete events, a so-called
chain of causation, is a more accurate way to describe disease.
Pathogenesis is a term used to describe the mechanism and sequence
of key events typical of a disease or in a given patient with a disease.
A related term, pathophysiology, emphasizes alterations in normal
physiology caused by a disease.
By analogy to a river, “upstream” and “downstream” refer to
events that occur earlier or later in the chain of causation. Since
correlation does not imply causation, we must be careful when
describing a putative chain of disease causation that we do not
assume one event causes another when both events are temporally
distinct but are due to shared (or distinct), yet unknown,
“upstream” causes. Nevertheless, in reference to a particular event
in the disease (e.g., appearance of a sign or symptom, positive test
result, evidence of disease progression, or even death), I borrow
two terms from law, proximate cause and ultimate cause.
A proximate cause is an occurrence or entity most closely
responsible for the event of interest. For example, a proximate
cause of most myocardial infarctions (heart attacks) is rupture of
the fatty clot-forming material contained within an atherosclerotic
plaque into the lumen of a coronary artery, resulting in thrombus
formation, arterial lumen occlusion, and necrosis of myocardium
in the artery’s territory. A proximate cause of the signs and symp-
toms in many autoimmune diseases appears to be rare but crafty
populations of autoreactive lymphocytes that escape elimination by
the immune system, secreting proteins (e.g., TNF-alpha, IL17)
10 Keith A. Wharton Jr.
that drive progression of disease, wreaking havoc on various tissues
and organs.
An ultimate cause (also known as root cause) is a further
upstream event or entity “without which” the proximate cause or
downstream events would not occur: Without atherosclerosis of
coronary arteries, most myocardial infarcts would not occur, and
without a mutation in the CFTR gene, cystic fibrosis (or its forme
frustes) would not occur. Common ultimate causes are genetic
mutations, environmental exposures, infectious agents, and inju-
ries; without the inciting event, e.g., a mutation or exposure, the
disease would likely not occur. For many diseases, ultimate causes
remain elusive.
While ordering events in disease pathogenesis can be a satisfy-
ing exercise, it is often said that “life itself is a terminal disease,”
implying that for every event, further upstream causes exist. For
example, origins of atherosclerosis remain under intense investiga-
tion, with evidence that genetic predisposition, environmental
exposure (e.g., diet), and perhaps the interaction between inoppor-
tune infections and the immune system, combine to create the
lesions of atherosclerosis [14]. There is epidemiologic evidence in
human, and experimental evidence in lower organisms, that paren-
tal and even grandparental environment can affect health in adult-
hood, possibly through epigenetic changes in DNA that are passed
through the germ line [15].
Indeed, the term chain of causation implies a linear order of
events in disease pathogenesis that for many diseases is inaccurate;
there are frequently nodes of divergence and convergence of events to
suggest a web or network of causation is a more apt term. For
example, “congestive heart failure” has secondary consequences
in other organ systems due to sluggish, compromised blood flow,
reduced oxygen delivery to tissues, and compensatory (and eventu-
ally decompensatory) hemodynamic changes—an example of diver-
gence. Many causes of chronic lung injury ultimately manifest as a
common histopathological picture of interstitial pneumonitis—an
example of convergence. Diseases with a common ultimate cause,
e.g., an inherited genetic mutation, can manifest differently even in
identical twins, highlighting the crucial interaction between genes,
environment, and other unknowns (epigenetics, serendipity) that
contribute to the heterogeneous nature of nearly all diseases.
Within and between categories of disease, two additional con-
cepts deserve mention, lumping and splitting. Lumping occurs
when distinct diseases are shown to share a common or related
pathogenesis or therapeutic vulnerability. There are several recent
notable examples of lumping: growth of two distinct types of
cancer, chronic myeloid leukemia (CML) and gastrointestinal
stromal tumor (GIST), is driven by overactivity of a shared intracel-
lular growth promoting protein kinase cascade, due to mutations
in related receptors that are susceptible to common kinase
Histopathology: A Canvas and Landscape of Disease in Drug and Diagnostic Development 11
inhibitors [16]. Splitting occurs when a disease that appears
homogeneous by traditional (often histopathological) criteria but
manifests heterogeneously in a diverse population is subdivided
into discrete diagnostic categories, typically by insights gained
from new technologies. One example of splitting is a type of cancer
termed diffuse large B-cell lymphoma (DLBCL). Although a given
patient’s prognosis with DLBCL bears some relationship to tradi-
tional, clinically measurable attributes such as tumor size, extent of
spread throughout the body, and symptoms, most DLBCLs are
indistinguishable from one another by histopathology: they consist
of abnormal appearing lymphoid tumor cells mixed with host
inflammatory cells. Expression profilingof mRNAs from a large
set of DLBCL tumor tissues revealed at least two subtypes with
mRNA expression profiles that bear some resemblance to benign B
lymphocytes at different developmental stages—the germinal B-cell
(GBC) type and activated B-cell (ABC) type [17]. Splitting is
important, because the GBC and ABC tumor types probably have
distinct ultimate causes (i.e., “driver” events such as gene mutations
that give rise to, or propagate, the disease), prognoses, and, pre-
sumably, therapeutic vulnerabilities that are under investigation.
The right degree of splitting for a given disease is the essence of
“personalized medicine.”
Events that occur on a particular space or time scale in disease
chain of causation can influence events that occur at vastly different
space or time scales. Consequently, understanding chain of causa-
tion can be a multidisciplinary pursuit, spanning epidemiology and
public health to molecular biophysics and electrophysiology.
The “channelopathies,” a heterogeneous group of diseases due to
dysfunction of a variety of ion channels, are excellent examples of
this principle [18]. Genetic mutation or acquired alteration in the
hERG potassium channel subunit alters cardiac conduction that
can manifest as chronically inefficient pumping of blood, or, in
rare cases, sudden death [19]. Another class of diseases, character-
ized by alterations in extracellular matrix proteins such as fibrillin,
causes give rise to mechanical fragility of blood vessels at sites of
high shear stress with loss of vascular tone, followed by high blood
pressure and the often lethal consequence of aortic dissection [20].
Feedback can be important to understand chain of causation in
disease. Feedback normally acts at critical upstream or downstream
“nodes” (control points) to amplify or dampen the activity of
biological systems. Feedback maintains homeostasis and reversibly
adapts a system to acute, repetitive, and chronic perturbations.
Proteins, molecular machines and pathways, cells, organ systems,
and our bodies as a whole have evolved a variety of homeostatic
mechanisms to optimize physiology as we interact with our envi-
ronment through our life cycle. Enzyme production and activity are
modified in response to metabolic demand; inflammation escalates
upon pathogen contact and then regresses upon clearance of the
12 Keith A. Wharton Jr.
invader; and blood pressure rises with arousal then falls with
perceived safety—all examples of feedback. Some types of feedback
occur within seconds, others over years. In disease, altered feedback
can be considered an ultimate and/or proximate cause of disease,
and can lead to specific signs and symptoms. Understanding how
feedback mechanisms are altered in disease can be crucial to devel-
oping effective targeting strategies, anticipating mechanisms of
resistance to therapies, and managing side effects.
Another concept widely invoked in normal physiology and
disease is crosstalk: how one defined molecular pathway influences
the activity of other pathways. While crosstalk in electronics is
generally undesirable, crosstalk between biological pathways
enables system integration. Altered inputs and outputs of different
molecular pathways are features of many diseases, leading to altera-
tions in cell function, tissue composition, and organ physiology,
and so opposition of pathway alterations is a common strategy to
target disease. Metabolic pathways that provide fundamental nutri-
ents such as glucose and fatty and amino acids to cells and tissues of
the body are among the most “crosstalked” pathways, in part
because so many factors must be considered when allocating cellu-
lar resources to energy utilization vs. storage. Diabetes is an exam-
ple of a disease with altered feedback and crosstalk. In its common
forms, it is characterized by failure to regulate serum glucose due to
a deficit of, or altered responses to, insulin. Multiple inputs regulate
insulin production and release from beta cells in the pancreatic
islets, and the effect of circulating insulin in different cell types
depends on inputs from other pathways. Crosstalk can be mediated
by a direct physical interaction between two components of simul-
taneously active yet distinct signaling pathways, and the conse-
quences of the interaction can be inhibitory or synergistic.
Understanding at what “level” crosstalk between two pathways
occurs in health or diseases, including the cell types and subcellular
compartments where key interactions occur, can inform diagnostic
and therapeutic strategies.
8 The Significance of the Lesion
If we must identify abnormalities in cells or their products to
declare “disease,” then it becomes necessary to pinpoint, visualize,
describe, and eventually measure the abnormality, known as the
lesion. In pathology circles, the lesion is a focus of thought about
disease. Intuitively, a lesion is mass or lump visible to the naked eye,
but it can also be microscopic (i.e., visible only with a microscope)
or molecular (e.g., a gene mutation is a genetic “lesion”). In
toxicology, the lesion is the histopathological change in tissue
structure and cell composition induced by exogenous agents, and
whether it is reversible or not—i.e., whether it resolves a suitable
Histopathology: A Canvas and Landscape of Disease in Drug and Diagnostic Development 13
time after the drug is withdrawn—often predicts whether a drug
can be safely administered to humans. Until we have “tricorders”
(a la Star Trek) capable of disease diagnosis at a distance, in most
cases it is only possible to study the nature of lesions in live patients
by studying diseased tissue, a fluid sample containing analytes
(molecules that can be identified and measured) that originated
from the diseased tissue, or another tissue that is specifically altered
as a consequence of the diseased cells or tissues. Beyond routine
histopathologic examination, attempts to understand lesions in
diseased tissue must pay strict attention to tissue procurement,
handling, and processing in order to preserve any molecules of
interest (i.e., the analytes to be measured). Lesions, or, properly
stated, visual representations of lesions, can be monitored in live
patients by a variety of imaging modalities including CT, MRI,
PET, SPECT, etc. Identifying and characterizing culprit lesions in
disease are key to understand chain of causation/pathogenesis as
well as to devise therapeutic strategies.
Although a lesion can be a variety of sizes, Virchow—the
“Father of Microscopic Pathology” who advocated for microscopy
in disease diagnosis—espouses the central role of the cell in disease
pathogenesis in his nineteenth century Pathology text:
. . .the chief point in this application of histology to pathology is to obtain
recognition of the fact, that the cell is really the ultimate morphological
element in which there is any manifestation of life, and we must not transfer
the seat of action to any point beyond the cell. [21]
Virchow’s quote is simultaneously prescient and timeless: he
implies efforts to understand each disease should focus on the
structure and function of cells. Thus, for each disease, the key
questions become: Which cells are diseased? What abnormalities
exist within the diseased cells? How do diseased cells affect nearby
cells to create the manifestations of disease? How can we detect the
disease with a diagnostic test? How might we design therapies that
reverse or mitigate disease-associated abnormalities in diseased tissues
(efficacy) with acceptable consequences for nondiseased tissues (safety/
toxicity)?
9 Heterogeneity of Disease
Medical textbooks impart the illusion to students (and the lay
public with unlimited internet access) that diseases, as they com-
mence in patients, tidily fit into categories. Experienced clinicians
know better, attesting to the unpredictable and heterogeneous

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